Christiane Nishibe

Detection of Mycobacterium bovis in bovine and bubaline tissues using nested-PCR for TbD1.

In the present study, a nested-PCR system, targeting the TbD1 region, involving the performance of conventional PCR followed by real-time PCR, was developed to detect Mycobacterium bovis in bovine/bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. In terms of analytical sensitivity, the DNA of M. bovis AN5 was detected up to 1.56 ng with conventional PCR, 97.6 pg with real-time PCR, and 1.53 pg with nested-PCR in the reaction mixture. The nested-PCR exhibited 100% analytical specificity for M. bovis when tested with the DNA of reference strains of environmental mycobacteria and closely-related Actinomycetales. A clinical sensitivity value of 76.0% was detected with tissue samples from animals that exhibited positive results in the comparative intradermal tuberculin test (CITT), as well as from those with lesions compatible with tuberculosis (LCT) that rendered positive cultures. A clinical specificity value of 100% was detected with tissue samples from animals with CITT- results, with no visible lesions (NVL) and negative cultures. No significant differences were found between the nested-PCR and culture in terms of detecting CITT+ animals with LCT or with NVL. No significant differences were recorded in the detection of CITT- animals with NVL. However, nested-PCR detected a significantly higher number of positive animals than the culture in the group of animals exhibiting LCT with no previous records of CITT. The use of the nested-PCR assay to detect M. bovis in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

Modulation of the immune response by Fonsecaea pedrosoi morphotypes in the course of experimental chromoblastomycosis and their role on inflammatory response chronicity

A common theme across multiple fungal pathogens is their ability to impair the establishment of a protective immune response. Although early inflammation is beneficial in containing the infection, an uncontrolled inflammatory response is detrimental and may eventually oppose disease eradication. Chromoblastomycosis (CBM), a cutaneous and subcutaneous mycosis, caused by dematiaceous fungi, is capable of inducing a chronic inflammatory response. Muriform cells, the parasitic form of Fonsecaea pedrosoi, are highly prevalent in infected tissues, especially in long-standing lesions. In this study we show that hyphae and muriform cells are able to establish a murine CBM with skin lesions and histopathological aspects similar to that found in humans, with muriform cells being the most persistent fungal form, whereas mice infected with conidia do not reach the chronic phase of the disease. Moreover, in injured tissue the presence of hyphae and especially muriform cells, but not conidia, is correlated with intense production of pro-inflammatory cytokines in vivo. High-throughput RNA sequencing analysis (RNA-Seq) performed at early time points showed a strong up-regulation of genes related to fungal recognition, cell migration, inflammation, apoptosis and phagocytosis in macrophages exposed in vitro to muriform cells, but not conidia. We also demonstrate that only muriform cells required FcγR and Dectin-1 recognition to be internalized in vitro, and this is the main fungal form responsible for the intense inflammatory pattern observed in CBM, clarifying the chronic inflammatory reaction observed in most patients. Furthermore, our findings reveal two different fungal-host interaction strategies according to fungal morphotype, highlighting fungal dimorphism as an important key in understanding the bipolar nature of inflammatory response in fungal infections.

Draft Genome Sequence of Mycobacterium bovis 04-303, a Highly Virulent Strain from Argentina

ABSTRACT Mycobacterium bovis strain 04-303 was isolated from a wild boar living in a free-ranging field in Argentina. This work reports the draft genome sequence of this highly virulent strain and the genomic comparison of its major virulence-related genes with those of M. bovis strain AF2122/97 and Mycobacterium tuberculosis strain H37Rv.

Draft Genome Sequence of Mycobacterium bovis Strain AN5, Used for Production of Purified Protein Derivative.

ABSTRACT Mycobacterium bovis strain AN5 has been used to produce purified protein derivative (PPD) for the intradermal test for bovine tuberculosis since it was introduced in 1948. This work reports the draft genome sequence of M. bovis AN5, which is used for the production of bovine PPD in Brazil, as well as comparisons to other strains of M. bovis and Mycobacterium tuberculosis.

Patterns and Processes of Mycobacterium bovis Evolution Revealed by Phylogenomic Analyses

Mycobacterium bovis is an important animal pathogen worldwide that parasitizes wild and domesticated vertebrate livestock as well as humans. A comparison of the five M. bovis complete genomes from the United Kingdom, South Korea, Brazil, and the United States revealed four novel large-scale structural variations of at least 2,000 bp. A comparative phylogenomic study including 2,483 core genes of 38 taxa from eight countries showed conflicting phylogenetic signal among sites. By minimizing this effect, we obtained a tree that better agrees with sampling locality. Results supported a relatively basal position of African strains (all isolated from Homo sapiens), confirming that Africa was an important region for early diversification and that humans were one of the earliest hosts. Selection analyses revealed that functional categories such as “Lipid transport and metabolism,” “Cell cycle control, cell division, chromosome partitioning” and “Cell motility” were significant for the evolution of the group, besides other categories previously described, showing importance of genes associated with virulence and cholesterol metabolism in the evolution of M. bovis. PE/PPE genes, many of which are known to be associated with virulence, were major targets for large-scale polymorphisms, homologous recombination, and positive selection, evincing for the first time a plethora of evolutionary forces possibly contributing to differential adaptability in M. bovis. By assuming different priors, US strains originated and started to diversify around 150–5,210 ya. By further analyzing the largest set of US genomes to date (76 in total), obtained from 14 host species, we detected that hosts were not clustered in clades (except for a few cases), with some faster-evolving strains being detected, suggesting fast and ongoing reinfections across host species, and therefore, the possibility of new bovine tuberculosis outbreaks.

Experimental results of a coarse-grained parallel algorithm for spanning tree and connected components

Dehne et al. present a BSP/CGM algorithm for computing a spanning tree and the connected components of a graph, that requires O(log p) communication rounds, where p is the number of processors. It requires the solution of the Euler tour problem which in turn is based on the solution of the list ranking problem. In this paper we present experimental results of a parallel algorithm that does not depend on the solution of the Euler tour or the list ranking problem. The proposed algorithm has the practical advantage of avoiding the list ranking computation and is based on the integer sorting algorithm which can be implemented efficiently on the BSP/CGM model. We implemented the proposed algorithm on a Beowulf cluster and on a grid running the InteGrade middleware. We obtained encouraging albeit modest speedup on a small Beowulf cluster and expect good speedups on the grid for larger size graphs and clusters.

Efficient Parallel Implementations of Multiple Sequence Alignment using BSP/CGM Model

Multiple sequence alignment is a fundamental tool in bioinformatics, widely used for predicting protein structure and function, reconstructing phylogeny and several other biological sequence analyses. Because it is a NP-hard problem, several studies have been conducted to propose efficient methods to solve it. Based on the well-known approximate algorithm proposed by Gusfield [8], we present two parallel solutions for this problem using the BSP/CGM model, with MPI and CUDA implementations. The results show that the use of parallel processing allows the manipulation of more and larger sequences.